Advances in recombinant DNA technology coupled with advances in plant transformation and regeneration technology have made it possible to introduce new genetic material into plant cells, plants or plant tissue, thus introducing new traits, eg., phenotypes, that enhance the value of the plant or plant tissue. The present invention relates to the introduction into plants of genes encoding colonization or virulence antigens or parts thereof of pathogens which colonize on or invade through mucosal surfaces of animal species. The present invention also relates to production of such colonization or virulence antigen or parts thereof by the plants. The invention further relates to the use of plant matter containing such colonization or virulence antigen or parts thereof for the oral immunization of humans and other animals to inhibit infection of the animal or human by the pathogen.
A. General Overview of Infectious Diseases and Immunity PA0 B. General Overview of Plant Transformation PA0 1. Agrobacterium-mediated Transformation PA0 2. Direct Gene Transfer PA0 C. General Overview of Plant Regeneration PA0 D. Means For Inducing a Secretory Immune Response
Infectious diseases are becoming an increasing problem for both animal and human health. Gillespie J. et al., Infectious Diseases of Domestic Animals, Comstock Press, Ithaca, N.Y. (1981); Mandell, G. L. et al., Principles and Practices of Infectious Diseases, 2nd Ed., John Wiley and Sons, New York (1985). Diseases caused by bacterial pathogens are particularly troublesome due to the increase in antibiotic-resistant pathogens. Most pathogens enter on or through a mucosal surface, with the exception of the insect-borne pathogens or those which enter the body through a wound. The former pathogens include, but are not limited to, pathogenic species in the bacterial genera Actinomyces, Aeromonas, Bacillus, Bacteroides, Bordetella, Brucella, Campylobacter, Capnocytophaga, Clamydia, Clostridium, Corynebacterium, Eikenella, Erysipelothrix, Escherichia, Fusobacterium, Hemophilus, Klebsiella, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Pasteurella, Proteus, Pseudomonas, Rickettsia, Salmonella, Selenomonas, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, and Versinia, pathogenic viral strains from the groups Adenovirus, Coronavirus, Herpesvirus, Orthomyxovirus, Picornovirus, Poxvirus, Reovirus, Retrovirus, Rotavirus, pathogenic fungi from the genera Aspergillus, Blastomyces, Candida, Coccidiodes, Cryptococcus, Histoplasma and Phycomyces, and pathogenic parasites in the genera Eimeria, Entamoeba, Giardia, and Trichomonas. It is generally acknowledged that prevention of infectious diseases would be much more cost-effective than attempts to treat infections once they occur. Thus, increased attention is being addressed to the development of vaccines for the effective immunization of humans and other animals. Germanier, R., Bacterial Vaccines, Academic Press, London (1984); Brown, F., Ann. Rev. Microbiol. 38, 221 (1984).
Animal and human hosts infected by a pathogen mount an immune response in an attempt to overcome the pathogen. There are three branches of the immune system: mucosal, humoral and cellular. Hood, L. E. et al., Immunology, 2 nd Ed., Benjamin Publishing Co., Menlo Park, Calif. (1984).
Mucosal immunity results from the production of secretory IgA (sIgA) antibodies in secretions that bathe all mucosal surfaces of the respiratory tract, gastrointestinal tract and the genitourinary tract and in secretions from all secretory glands. McGhee, J. R. et al.,Annals N.Y. Acad. Sci. 409, (1983). These sIgA antibodies act to prevent colonization of pathogens on a mucosal surface (Williams, R. C. et al., Science 177, 697 (1972); McNabb, P. C. et al., Ann. Rev. Microbiol. 35, 477 (1981) and thus act as a first line of defense to prevent colonization or invasion through a mucosal surface. The production of sIgA can be stimulated either by local immunization of the secretory gland or tissue or by presentation of an antigen to either the gut-associated lymphoid tissue (GALT or Peyer's patches) or the bronchial-associated lymphoid tissue (BALT). Cebra, J. J. et al., Cold Spring Harbor Symp. Quant. Biol. 41, 210 (1976); Bienenstock, J. M., Adv. Exp. Med. Biol. 107, 53 (1978); Weisz-Carrington, P. Et al., J. Immunol 123, 1705 (1979); McCaughan, G. et al., Internal Rev. Physiol 28, 131 (1983). Membranous microfold cells, otherwise known as M Cells, cover the surface of the GALT and BALT and may be associated with other secretory mucosal surfaces. M cells act to sample antigens from the luminal space adjacent to the mucosal surface and transfer such antigens to antigen-presenting cells (dendritic cells and macrophages), which in turn present the antigen to a T lymphocyte (in the case of T-dependent antigens), which process the antigen for presentation to a committed B cell. B cells are then stimulated to proliferate, migrate and ultimately be transformed into an antibody-secreting plasma cell producing IgA against the presented antigen. When the antigen is taken up by M cells overlying the GALT and BALT, a generalized mucosal immunity results with sIgA against the antigen being produced by all secretory tissues in the body. Cebra et al., supra; Bienenstock et al., supra; Weinz-Carrington et al., supra; McCaughan et al., supra. Oral immunization is therefore a most important route to stimulate a generalized mucosal immune response and, in addition, leads to local stimulation of a secretory immune response in the oral cavity and in the gastrointestinal tract.
Humoral immunity results from production of IgG and IgM in serum and potentiates phagocytosis of pathogens, the neutralization of viruses, or complement-mediated cytotoxicity of pathogens (Hood et al., supra). The immunity to a pathogen can be transmitted from the mother to the offspring in both birds and mammals by delivery of the secretory antibody either in the egg or in the colostrum or by placental transfer of serum antibody in the case of mammals. McGhee et al., supra, McNabb et al., supra; Mestecky, J., J. Clin. Immunol., 7, 265 (1987).
Cellular immunity is of two types: One is termed a delayed-type hypersensitivity response which causes T lymphocytes to stimulate macrophages to kill bacterial, parasitic, and mycotic pathogens. In the other type, cytotoxic T lymphocytes are directed to kill host cells infected with viruses. Hood, et al. supra.
Secretory IgA antibodies directly inhibit the adherence of microorganisms to mucosal epithelial cells and to the teeth of the host. Abraham, S. N. et al., Advances in Host Defense Mechanisms, Raven Press, N.Y., 4, 63 (1985). Liljemark, W. F. et al., Infect. Immun. 26, 1104 (1979). Reinholdt, J. et al., J. Dent. Res. 66, 492 (1987). This may be done by agglutination of microorganisms, reduction of hydrophobicity, Magnusson, K. E., et al., Immunology 36, 439 (1979), or negative charge and blockage of microbial adhesions. These anti-adherence effects are amplified by other factors such as secretory glycoproteins, continuous desquamation of surface epithelium and floral competition. Abraham, S. N. et al., supra. Shedlofsky, S. et al., J. Infect. Dis. 129, 296 (1974). For example, oral immunization against inactivated Vibrio cholerae to induce a secretory immune response results in a 10-to 30- fold decrease in intestinal numbers.
Clinical experience with human peroral poliovirus vaccine and several peroral or intranasal virus vaccines applied in veterinary medicine shows that sIgA plays a decisive role in protective effect by the mucosal immune system against respiratory and enteric viral infections. Rusel-Jones, G. J. et al., Int. Arch. Allergy Appl. Immunol. 66, 316 (1981). Ogra, P. L. et al., In J. Bienenstock (ed), Immunology of the Lung and Upper Respiratory Tract. McGraw-Hill, N.Y. 242 (1984). The effect of sIgA appears to be that of inhibiting the entry of viruses into host cells rather than prevention of attachment. Taylor, H. P. et al., J. Exp. Med. 161, 198 (1985). Kilian, M. et al., Microbiol. Rev. 52, 296 (1988).
Various methods are known in the art to accomplish the genetic transformation of plants and plant tissues (i.e., the stable introduction of foreign DNA into plants). These include transformation by Agrobacterium species and transformation by direct gene transfer.
A. tumefaciens is the etiologic agent of crown gall, a disease of a wide range of dicotyledons and gymnosperms, DeCleene, M. et al., Bot. Rev. 42, 389 (1976), that results in the formation of tumors or galls in plant tissue at the site of infection. Agrobacterium, which normally infects the plant at wound sites, carries a large extrachromosomal element called the Ti (tumor-inducing) plasmid.
Ti plasmids contain two regions required for tumorigenicity. One region is the T-DNA (transferred-DNA) which is the DNA sequence that is ultimately found stably transferred to plant genomic DNA. The other region required for tumorigenicity is the vir (virulence) region which has been implicated in the transfer mechanism. Although the vir region is absolutely required for stable transformation, the vir DNA is not actually transferred to the infected plant. Chilton, M-D. et al., Cell 11, 263 (1977), Thomashow, M. F. et al., Cell 19, 729 (1980). Transformation of plant cells mediated by infection with A. tumefaciens and subsequent transfer of the T-DNA alone have been well documented. See, for example, Bevan, M. W. et al., Int. Rev. Genet. 16, 357 (1982).
After several years of intense research in many laboratories, the Agrobacterium system has been developed to permit routine transformation of a variety of plant tissue. See, for example, Schell, J. et al., Bio/Technology 1, 175 (1983); Chilton, M-D, Scientific American 248, 50 (1983). Representative tissues transformed in this manner include tobacco, Barton, K. A. et al., Cell 32, 1033 (1983); tomato, Fillatti, J. et al., Bio/Technology 5, 726 (1987); sunflower, Everett, N. P. et al., Bio/Technology 5, 1201 (1987); cotton, Umbeck, P. et al., Bio/Technology 5, 263 (1987); rapeseed, Pua, E. C. et al., Bio/Technology 5, 815 (1987); potato, Facciotti D. et al., Bio/Technology 3, 241 (1985); poplar, Pythoud, F. et al., Bio/Technology 5, 1323 (1987); and soybean, Hinchee, M. A. et al., Bio/Technology 6, 915 (1988).
Agrobacterium rhizogenes has also been used as a vector for plant transformation. That bacterium, which incites root hair formation in many dicotyledonous plant species, carries a large extrachromosomal element called an Ri (root-inducing) plasmid which functions in a manner analogous to the Ti plasmid of A. tumefaciens. Transformation using A. rhizogenes has developed analogously to that of A. tumefaciens and has been successfully utilized to transform, for example, alfalfa, Sukhapinda, K. et al., Plant Mol. Biol. 8, 209 (1987); Solanum nigrum L., Wei Z-H, et al., Plant Cell Reports 5, 93(1986); and, poplar, Pythoud, et al., supra.
Several so-called direct gene transfer procedures have been developed to transform plants and plant tissues without the use of an Agrobacterium intermediate. In the direct transformation of protoplasts the uptake of exogenous genetic material into a protoplast may be enhanced by use of a chemical agent or electric field. The exogenous material may then be integrated into the nuclear genome. The early work was conducted in the dicot Nicotiana tabacum (tobacco) where it was shown that the foreign DNA was incorporated and transmitted to progeny plants. Paszkowski, J. et al., EMBO J, 3, 2717 (1984); and Potrykus, I. et al., Mol. Gen. Genet. 199, 169 (1985).
Monocot protoplasts have also been transformed by this procedure: for example, Triticum monococcum, Lorz H. et al., Mol. Gen. Genet. 199, 178 (1985); Lolium multiflorum (Italian ryegrass), Potrykus, I. et al., Mol. Gen. Genet 199, 183 (1985); maize, Rhodes, C., et al., Bio/Technology 5, 56 (1988); and Black Mexican sweet corn, Fromm, M. et al., Nature 319, 791 (1986).
Introduction of DNA into protoplasts of N. tabacum is effected by treatment of the protoplasts with an electric pulse in the presence of the appropriate DNA in a process called electroporation. Fromm, M. E., in Methods in Enzymology, eds. Wu, R. and Grossman, L., Academic Press, Orlando, Fla., Volume 153, 307 (1987) and Shillito, R. D. and Potrykus, I. in Methods in Enzymology, eds., Wu, R. and Grossman, L., Academic Press, Orlando, Fla. Volume 153, 283 (1987). Protoplasts are isolated and suspended in a mannitol solution. Supercoiled or circular plasmid DNA is added. The solution is mixed and subjected to a pulse of about 400 Vcm at room temperature for less than 10 to 100 .mu. sec. A reversible physical breakdown of the membrane occurs to permit DNA uptake into the protoplasts.
DNA viruses have been used as gene vectors. A cauliflower mosaic virus carrying a modified bacterial methotrexate-resistance gene was used to infect a plant. The foreign gene was systematically spread in the plant. Brisson, N. et al., Nature 310, 511 (1984). The advantages of this system are the ease of infection, systematic spread within the plant, and multiple copies of the gene per cell.
Liposome fusion has also been shown to be a method for transformation of plant cells. Protoplasts are brought together with liposomes carrying the desired gene. As membranes merge, the foreign gene is transferred to the protoplast. Dehayes, A. et al., EMBO J. 4, 2731 (1985).
Polyethylene glycol (PEG) mediated transformation has been carried out in N. tabacum a dicot, and Lolium multiflorum, a monocot. It is a chemical procedure of direct gene transfer based on synergistic interaction between Mg.sup.2+, PEG, and possibly Ca.sup.2+. Negrutiu, R. et al., Plant Mol. Biol. 8, 363 (1987).
Alternatively, exogenous DNA can be introduced into cells or protoplasts by microinjection. A solution of plasmid DNA is injected directly into the cell with a finely pulled glass needle. In this manner, alfalfa protoplasts have been transformed by a variety of plasmids, Reich, T. J. et al., Bio/Technology 4, 1001 (1986).
A more recently developed procedure for direct gene transfer involves bombardment of cells by microprojectiles carrying DNA. Klein, T. M. et al., Nature 327, 70 (1987). In this procedure called particle acceleration, tungsten or gold particles coated with the exogenous DNA are accelerated toward the target cells. At least transient expression has been achieved in onion. This procedure has been utilized to introduce DNA into Black Mexican sweet corn cells in suspension culture and maize immature embryos and also into soybean protoplasts. Klein, T. M. et al., Bio/Technology 6, 559 (1988). McCabe, D. E. et al., Bio/Technology 6, 923 (1988). Stably transformed cultures of maize and tobacco have been obtained by microprojectile bombardment. Klein, T. M. et al (1988), supra. Stably transformed soybean plants have been obtained by this procedure. McCabe, D. E. et al., supra.
Just as there are a variety of methods for the transformation of plant tissue, there are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. In recent years, it has become possible to regenerate many species of plants from callus tissue derived from plant explants. The plants which can be regenerated from callus include monocots, such as corn, rice, barley, wheat and rye, and dicots, such as sunflower, soybean, cotton, rapeseed and tobacco.
Regeneration of plants from tissue transformed with A. tumefaciens has been demonstrated for several species of plants. These include sunflower, Everett, N. P. et al., supra; tomato, Fillatti, J. J. et al., supra; white clover, White, D. W. R. et al., Plant Mol. Biol. 8, 461 (1987); rapeseed, Pua, E-C. et al., supra; cotton, Umbeck, P. et al., supra; tobacco, Horsch, R. B. et al., Science 225, 1229 (1985) and Hererra-Estrella, L. et al., Nature 303, 209 (1983); and poplar, Pythoud et al., supra. The regeneration of alfalfa from tissue transformed with A. rhizogenes has been demonstrated by Sukhapinda, K. et al., supra.
Plant regeneration from protoplasts is a particularly useful technique. See Evans, D. A. et al., Handbook of Plant Cell Culture 1, 124 (1983). When a plant species can be regenerated from protoplasts, then direct gene transfer procedures can be utilized, and transformation is not dependent on the use of A. tumefaciens. Regeneration of plants from protoplasts has been demonstrated for rice, Abdullah, R. et al., Bio/Technology 4, 1087 (1987); tobacco, Potrykus, I. et al., supra; rapeseed, Kansha, et al., Plant Cell Reports 5, 101 (1986); potato, Tavazza, R. et al., Plant Cell Reports 5, 243 (1986); eggplant, Sihackaki, D. et al., Plant Cell, Tissue, Organ Culture 11, 179 (1987); cucumber, Jia S-R,. et al., J. Plant Physiol. 124, 393 (1986); poplar, Russel, J. A. et al., Plant Sci. 46, 133 (1986); corn, Rhodes, C. et al., supra; and soybean, McCabe, D. E. et al., supra.
The M cells overlying the Peyer's patches of the gut-associated lymphoid tissue (GALT) are capable of taking up a diversity of antigenic material and particles (Sneller, M. C. and Strober, W., J. Inf. Dis. 154, 737 (1986). Because of their abilities to take up latex and polystyrene spheres, charcoal, microcapsules and other soluble and particulate matter, it is possible to deliver a diversity of materials to the GALT independent of any specific adhesive-type property of the material to be delivered. In this case, antigen delivery to the GALT leads to a generalized mucosal immune response with sIgA production against the antigen on all mucosal surfaces and by all secretory glands. One can also stimulate a local secretory immune response by antigen delivery to a mucosal surface or to a secretory gland. The mechanism(s) for generating such a localized secretory immune response is(are) poorly understood. Recent evidence, Black, R. E. et al. Infect. Immun. 55,1116 (1987); Elson, C. O., in Curr. Top. Microbiol. Immunol. 146, 29 (1989), indicate that the B subunit of cholera toxin when administered orally with an antigen serves as an adjuvant to enhance the protective immune response. It therefore follows, since the B subunit of cholera toxin as well as of the E. coli heat-labile enterotoxin are capable of attaching to the GM-1 ganglioside of the intestinal epithelium and causing translocation across the epithelial membrane, that such pilot or targeting proteins might be important in eliciting a local secretory immune response.
It is of course possible to consider fusing a gene for a given colonization and/or virulence antigen to an N-terminal or C-terminal sequence specifying the B subunit of cholera toxin, the B subunit of heat-labile enterotoxin, Yamamoto, T. et al. J. Biol. Chem. 259, 5037 (1984), the PapG protein adhesion that specifically binds to .alpha.-D-galactopyranosyl-(1,4)-.beta.-D-galactopyranoside, Lund, B. et al., Proc. Natl. Acad. Sci. USA 84, 5898 (1987), or the invasions causing penetration of bacteria through epithelial cell membranes as identified in and cloned from Yersinia pseudotuberculosis, Isberg, R. R., et al. Cell 50, 769 (1987), Shigella and Salmonella. Galan, J. et al., Poc. Natl. Acad. Sci. U.S.A. 86, 6383 (1989); Curtiss, R. III et al., in Curr. Top. Microbiol. Immunol. 146, 35 (1989). In each case, it can be anticipated that the product of the gene fusion will be more readily transported into cells of the intestinal mucosa and lead to enhanced local secretory immune responses. It is also possible that this form of gene fusion would facilitate uptake and presentation of antigens to the GALT. The production of sIgA against a particular antigen can also be further enhanced by the addition of orally-administered adjuvants, such as microbial cell wall constituents Michalek, S. M. et al., in Curr. Top. Microbiol. Immunol. 146, 51 (1989).
It is therefore evident that stimulation of a specific sIgA respone of a both local and generalized nature can be achieved by oral immunization with purified proteins, Taubman, M. A. and D. J. Smith, in Curr. Top. Microbiol. Immunol. 146, 187 (1989), microencapsulated microbial products and viruses, Eldridge, J. H. et al., in Curr. Top. Microbiol. Immunol. 146, 59 (1989), whole-killed bacteria, Michalek et al., Science 191, 1238 (1976), and by ingestion of live attenuated viruses, Cebra, et al., supra, and bacteria, Curtiss, R. III et al., in Proceedings of the Tenth International Convocation on Immunology, 261. H. Kohler et al., Eds., Longman Scientific and Technical, Harlon, Essex, Great Britain (1987). The relative importance of the secretory immune system becomes apparent when one realizes that 80% of the antibody-secreting cells in the body produce sIgA and that twice as much sIgA is secreted into the gastrointestinal tract than IgG is produced to enter the circulatory system each day, Brandtzaeg, P., in Curr. Top. Microbiol. Immunol. 146, 13 (1989).
The Streptococcus mutans group of microorganisms constitute the principal etiologic agents of dental caries. Gibbons, R. J. et al., Ann. Rev. Med. 26, 121 (1975); Hamada, S. et al., Microbiol. Rev. 44, 331 (1980). They colonize the tooth surface and remain there throughout life. Oral ingestion of killed S. mutans leads to the production of sIgA against S. mutans antigens in saliva, Michalek, S. M. et al., Science 191, 1238 (1976); Mastecky, J. et al., J. Clin. Invest. 61, 731 (1978) and this has been shown to be effective in preventing S. mutans colonization on the teeth of rodents and primates and thereby prevent induction of caries. Michalek et al., supra; Challacombe, S. J. et al., Arch. Oral Biol. 24, 917 (1980). Since sIgA must be present prior to colonization to be effective, individuals immunized to produce sIgA against S. mutans colonization antigens after colonization has occurred will continue to be colonized with S. mutans unless the bacteria are mechanically removed during dental propylaxis. Curtiss, R. III, in Curr. Top. Microbiol. Immunol. 118, 253 (1985). A diversity of techniques are used to determine which surface constituents of a pathogen are important for colonization and expression of virulence by that pathogen. Thus mutants can be isolated and tested for ability to colonize or cause disease. Gene cloning can be used to produce a gene product in a heterologous microorganism. The expressed gene product can be used to immunize animals to see whether colonization and/or virulence by the pathogen is inhibited. Based on such studies, scientists can infer relative importance to various colonization and virulence antigens and thereby choose those that are appropriate to use in vaccine compositions so as to immunize human or other animal hosts and prevent colonization and infection by the pathogen. Such studies have been performed with the S. mutans group of microorganisms to demonstrate the critical importance of the surface protein antigen A (SpaA; also known as antigen I/II, B, and P1), glucosyltransferases, dextranase and glucan-binding proteins. Curtiss, 1985 supra.
The surface protein antigen A (SpaA) constitutes a major protein antigen on the surface of S. mutans. Curtiss, R. III, et al., in Streptococcal Genetics, Ferretti, J. J. et al., Ed., American Society for Microbiology, Washington, D.C. pp. 212-216 (1987). The spaA gene has been cloned, Holt, R. G. et al., Infect. Immun. 38, 147 (1982), partially sequenced and the major antigenic determinants mapped. It is known that mice and humans intentionally or naturally immunized by oral ingestion of S. mutans produce sIgA in saliva against the SpaA protein. It is furthermore known that immunization of monkeys with antigen I/II (which is essentially immunologically identical to SpaA, Holt et al., supra) yields protective immunity against S. mutans colonization and S. mutans-induced dental caries, Russell, M. W. et al. Immunol. 40, 97 (1980).
Invasive Salmonella, such as S. typhimurium and S. typhi constitute the etiologic agents for typhoid fever in mice and humans, respectively. They gain access to deep tissues following oral ingestion by attaching to, invading, and proliferating in the GALT. Carter and Collins J. Exp. Med. 139, 1189 (1974). Salmonella can be rendered avirulent so as not to induce disease by introducing mutations in known genes. Germanier, R. et al., Infect. Immun. 4, 663 (1971); Germanier, R. et al., J. Infect. Dis. 131, 553 (1975); Hoiseth and Stocker, Nature 291, 238 (1981); Curtiss et al., Infect. Immun. 55, 3035 (1987). Such mutants are immunogenic when administered orally and retain their tissue tropism for the GALT. Curtiss, R. III, J. Dent. Res. 65, 1034 (1986); Curtiss, R. III et al., in Proceedings of the Tenth International Convocation on Immunology, 261. H. Kohler et al., Eds., Longman Scientific and Technical, Harlon, Essex, Great Britain (1987); Curtiss, R. III, et al., Infect. Immun. 55, 3035 (1987).
A number of S. typhimurium and S. typhi strains which possess various deletion mutations rendering them avirulent have been constructed with the ability to produce colonization and/or virulence antigens from several pathogens. Oral immunization leads to production of sIgA and IgG responses against the expressed antigen. Formal, S. B. et al., Infect. Immun. 34, 746 (1981); Stevenson, G. et al., FEMS Microbiol. Lett. 28, 317 (1985); Clements, J. D. et al., Infect. Immun. 53, 685 (1986); Maskell D. et al., in Vaccines 86, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 213-217 (1986). Recombinant avirulent Salmonella expressing the S. mutans SpaA and glucosyltransferase proteins have been constructed. Curtiss et al., in: The Secretory Immune System, J. R. McGhee and J. Mestecky, Eds., Ann. N.Y. Acad. Sci. 409, 688 (1983); Curtiss supra (1986); Curtiss et al., supra (1987); Curtiss et al., Vaccine 6, 155 (1988). Secretory antibodies (sIgA) against SpaA have been produced in saliva following oral immunization with avirulent Salmonella strains expressing the S. mutans SpaA protein, Curtiss, R. III et al., in Mol. Microbiol. Immunol of Streptococcus mutans, Hamada, S. et al., Eds., Elsevier, N.Y. pp. 173-180 (1986); Katz, J. et al., in Recent Advances In Mucosal Immunology, Part B, Mestecky, J. et al., Ed., Plenum Publishing Corp., pp. 1741-1747 (1987); Curtiss et al. 1987, supra.